A plasmin inhibitory peptide derived from β-lactoglobulin, its screening method and application

Abstract

This invention belongs to the field of bioactive peptide technology, and relates to a plasmin inhibitory peptide derived from β-lactoglobulin, its screening method, and its application. Its amino acid sequence is shown in SEQ ID NO.1. This invention selects β-lactoglobulin from bovine milk as the source of the plasmin inhibitory peptide. Using the structure of the naturally conformated μ-plasmin as a template, molecular docking is performed between β-lactoglobulin and plasmin. Molecular dynamics simulations are conducted on the complex until equilibrium is reached. Based on the interaction between the active site of plasmin and the β-lactoglobulin sequence, targeted screening is performed on polypeptide sequences within the β-lactoglobulin sequence that may have inhibitory activity, resulting in a small, short peptide that specifically inhibits plasmin and can be rapidly synthesized artificially. Adding this plasmin inhibitory peptide to UHT milk increases the stability of sterilized milk and extends its shelf life, showing promising application prospects.

Description

Technical Field

[0001] This invention belongs to the field of bioactive peptide technology, specifically relating to a plasmin inhibitory peptide derived from β-lactoglobulin, its screening method, and its application. Background Technology

[0002] Ultra-high temperature (UHT) milk is a product of fresh milk sterilized through direct or indirect heating and can be stored at room temperature for 4-6 months. However, heat-resistant endogenous enzymes in milk can cause UHT milk to deteriorate during storage, especially fibrinolytic enzymes that cause casein hydrolysis, leading to bitterness, precipitation, and gelation, severely affecting the shelf life of UHT milk, particularly direct UHT milk. Therefore, reducing the activity of heat-resistant endogenous fibrinolytic enzymes is an urgent problem to be solved in order to extend the shelf life of UHT milk.

[0003] Current applications of plasmin inhibitors are primarily clinical, aimed at reducing postoperative bleeding, and have not yet been applied to dairy products. Developing high-affinity plasmin inhibitors for use in dairy products to reduce plasmin activity in UHT-containing milk and extend its shelf life holds great promise. Currently, most naturally occurring plasmin inhibitors are serine protease inhibitors, lacking specific plasmin inhibitors. This is mainly due to the structural similarities between different serine proteases, making the design of specific plasmin inhibitors challenging. Furthermore, most existing protein or peptide inhibitors typically contain one or more complex Kunitz or Kazal domains, making them difficult to synthesize artificially. Summary of the Invention

[0004] The purpose of this invention is to fill the gap in the development of plasmin inhibitors in the food field and to solve the problems of cumbersome separation and extraction / difficult rapid synthesis of plasmin inhibitors. This invention proposes a plasmin inhibitory peptide derived from β-lactoglobulin, its screening method, and its application. Using the structure of the natural conformation of μ-plasmin as a template, and employing molecular docking, a small short peptide that specifically inhibits plasmin is extracted from the flexible loop structure of the β-lactoglobulin secondary structure. This is a plasmin inhibitory short peptide that can be rapidly synthesized artificially. Adding this peptide to fresh UHT milk successfully eliminated the unstable stratification phenomenon in milk caused by plasmin during storage, extending the shelf life of UHT milk.

[0005] The technical solution of this invention is:

[0006] This invention protects a β-lactoglobulin-derived plasmin inhibitory peptide, the amino acid sequence of which is shown in SEQ ID NO.1, EKTKIPAV.

[0007] This invention also protects a method for screening the β-lactoglobulin-derived plasmin inhibitory peptide, comprising the following steps:

[0008] (1) Use protein databases to obtain protein structure data of plasmin and β-lactoglobulin, complete molecular docking of plasmin and β-lactoglobulin, and perform 200 ns molecular dynamics simulation of the β-lactoglobulin and plasmin complex.

[0009] (2) Analyze the interaction interface between β-lactoglobulin and plasmin, and screen for polypeptide sequences in the β-lactoglobulin sequence that bind to the active site of plasmin; among them, the active site of plasmin is a triplet composed of 624HIS, 667ASP and 762SER.

[0010] (3) Based on the characteristic that the sequence of the plasmin inhibitory peptide is mainly composed of lysine and arginine at the P1 site, and combined with the amino acid sequence of β-lactoglobulin covering the plasmin active site, a suitable amino acid peptide segment is selected as the target peptide segment, and the target peptide segment is synthesized according to the sequence.

[0011] Furthermore, the plasmin inhibitory peptide is chemically synthesized by solid-phase synthesis and has a purity of ≥95%.

[0012] Furthermore, the plasmin inhibitory peptide contains 8 amino acid residues and has a molecular weight of 885.06 Da.

[0013] This invention also protects the use of the β-lactoglobulin-derived plasmin inhibitory peptide in the preparation of plasminase inhibitors.

[0014] The application of the β-lactoglobulin-derived plasmin inhibitory peptide in the preparation of food additives.

[0015] Furthermore, the food additive is used in dairy products, and is added to UHT milk as an additive to extend the shelf life of ultra-high temperature sterilized dairy products.

[0016] This invention protects a food additive whose active ingredient includes the aforementioned plasmin inhibitory peptide.

[0017] This invention protects the use of the β-lactoglobulin-derived plasmin inhibitory peptide in the preparation of hemostatic drugs.

[0018] The beneficial effects of this invention are:

[0019] (1) The plasmin inhibitory peptide provided by this invention is derived from β-lactoglobulin. This is the first study on plasmin inhibitory peptide derived from β-lactoglobulin. One plasmin inhibitory peptide was screened and obtained. This peptide contains 8 amino acids, has a simple short sequence structure, a clear mechanism of action, and can be synthesized rapidly, in large quantities, and at low cost, and has good application prospects.

[0020] (2) The screening method provided by the present invention selects β-lactoglobulin in bovine milk as the source of plasmin inhibitory peptides, uses the structure of μ-plasmin as a template, obtains the β-lactoglobulin and plasmin complex using molecular docking technology, and performs molecular dynamics simulation of the docked complex at room temperature to achieve equilibrium; by analyzing the amino acid sequence of β-lactoglobulin near the active site of plasmin, and combining the amino acid sequence information of existing plasmin inhibitors, targeted screening of polypeptide sequences in the β-lactoglobulin sequence that may have plasmin inhibitory activity is performed; based on the characteristics of the binding of polypeptide sequences to the active site of plasmin, the present invention can directly screen amino acid polypeptide segments with plasmin inhibitory activity from the β-lactoglobulin amino acid sequence without protein hydrolysis.

[0021] (3) The present invention can synthesize plasmin inhibitory peptides by solid-phase synthesis. Adding the peptides to dUHT milk improves its storage stability and extends its shelf life. It has broad application prospects and solves the lack of specific plasmin inhibitors in the food field. Attached Figure Description

[0022] Figure 1 This is a schematic diagram of the molecular docking and interaction interface between β-lactoglobulin and plasmin.

[0023] Figure 2 To simulate the changes in RMSD values ​​of β-lactoglobulin and plasmin over 200 ns using molecular dynamics;

[0024] Figure 3 This demonstrates the inhibitory effect of plasmin inhibitory peptide YHX-PIP-2 on plasmin.

[0025] Figure 4 The apparent changes of plasmin inhibitory peptide YHX-PIP-2 in dUHT milk with and without the addition of plasmin inhibitory peptide YHX-PIP-2 after storage at 37°C for one week;

[0026] Figure 5 The effect of adding plasmin inhibitory peptide YHX-PIP-2 on the TSI value of dUHT milk stability. Detailed Implementation

[0027] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0028] To further understand the present invention, it will be further described in conjunction with the accompanying drawings and embodiments.

[0029] Unless otherwise specified, the experimental methods and detection methods involved in the following embodiments are all conventional experimental methods and detection methods that already exist in the prior art.

[0030] Example 1: Molecular docking and molecular dynamics simulation of β-lactoglobulin and plasmin

[0031] The plasminogen structure data file (E1B726) and the β-lactoglobulin file (5IO6) were downloaded from the AlphaFoldprotein protein structure database and the RCSB Protein Data Bank protein database, respectively. In this invention, μ-plasmin (amino acid sequence 585-812), which contains an active site in the plasminogen structure, was selected as a template for the interaction between β-lactoglobulin and plasminogen. Rigid docking of plasminogen and β-lactoglobulin was performed using Z-DOCK (3.0.2) software.

[0032] The binding energy and interacting amino acids in the docking results were initially analyzed using PDBePISA (https: / / www.ebi.ac.uk / pdbe / pisa / ). The docking model with the lowest Gibbs free energy was selected (ΔG = -4.7 kcal / mol in this method). The model was then submitted to Haddock for further flexible docking, and the model with the highest score was selected as the object for molecular dynamics simulation. Molecular dynamics simulation was performed to ensure that the β-lactoglobulin and plasmin complex were in equilibrium, which is more realistic. A 200 ns molecular dynamics simulation of the β-lactoglobulin and plasmin complex was conducted using Gromacs software at 300 K, employing the amber99sb-ildn force field and selecting the tip3p water model to dissolve the complex in a dodecahedral box. 0.1 mol / L Na₂ was randomly inserted. + and Cl - Ions were used to neutralize the system's charge. Energy minimization was then performed. Constraint pre-equilibrations of 200 ps NVT and 200 ps NPT were then conducted to bring the equilibrium temperature to 298 K. Berendsen thermostat and Parrinello-Rahman pressure were used to maintain the temperature and pressure, respectively. All nonbonded interactions, including Coulomb and van der Waals potentials, were represented using... Verlet cutoff mode. Electrostatic interactions were handled using the Particle Mesh Ewald (PME) summation method. Finally, position constraints were removed, and the system was simulated with a trajectory time of 200 ns.

[0033] like Figure 2As shown, after a 200 ns molecular dynamics simulation, the RMSD of both β-lactoglobulin and plasmin monomers fluctuated around 0.2 nm, indicating that the 200 ns molecular dynamics simulation had brought the β-lactoglobulin and plasmin complex to an equilibrium state.

[0034] Example 2: Screening for potential plasmin inhibitory peptides in β-lactoglobulin sequences

[0035] Plasmin, as a serine protease, has an active site consisting of a triplet of 624HIS, 667ASP, and 762SER. For example... Figure 1 As shown, after the β-lactoglobulin and plasmin complex reach equilibrium, the active site of plasmin is partially covered by the β-lactoglobulin sequence. Analysis of existing plasmin inhibitory peptide sequences revealed that they are predominantly lysine (Lys) and contain a small amount of arginine (Arg) at the P1 site. Combined with the amino acid sequence of β-lactoglobulin covering the plasmin active site, an octapeptide sequence was screened as a potential plasmin inhibitory peptide, YHX-PIP-2, with the amino acid sequence EKTKIPAV (SEQ ID NO. 1).

[0036] Example 3: Peptide Synthesis and Activity Verification

[0037] The selected peptide YHX-PIP-2 was synthesized using a solid-phase peptide synthesis technique. The synthesized peptide EKTKIPAV exhibited good water solubility. The effect of the plasmin inhibitory peptide YHX-PIP-2 on plasmin activity was assessed using a substrate-based colorimetric method. The plasmin inhibitory peptide YHX-PIP-2 was dissolved in 0.01M phosphate buffer (pH 7.4) to prepare a 1 mg / mL peptide solution. 50 μL of this peptide solution was mixed with 10 μL of 0.1 U / mL plasmin, followed by the addition of 140 μL of 0.1 mM D-Val-Leu-Lys p-nitroaniline dihydrochloride. The absorbance was immediately measured at 405 nm using a microplate reader. After shaking for three seconds, measurements were taken every 5 minutes, maintaining the temperature at 37℃.

[0038] A blank control group was set up. The blank control group sample differed from the β-lactoglobulin group (YHX-PIP-2) sample in that the peptide solution in the blank control group sample was replaced with phosphate buffer, and the absorbance at 405 nm was measured. The results are as follows: Figure 3 As shown, the addition of peptide YHX-PIP-2 can reduce the ability of plasmin to hydrolyze substrates, indicating that the plasmin inhibitory peptide YHX-PIP-2 extracted from β-lactoglobulin has the function of inhibiting plasmin activity.

[0039] Example 4: Application of plasmin inhibitory peptide in improving the stability of dUHT milk

[0040] Fresh direct ultra-high temperature (dUHT) sterilized milk is prone to aging and gelation caused by plasmin. Therefore, the effect of peptide YHX-PIP-2 on the shelf life of milk was verified using dUHT-sterilized milk. The heat treatment conditions for fresh dUHT-sterilized milk were 153℃ for 0.25s. After sterilization, 23 mL of fresh dUHT-sterilized milk was added to a dedicated transparent glass vial for Turbiscan stability analysis as a blank control. In addition, 300 μL of 8 mM peptide YHX-PIP-2 (0.1 M PBS, pH 7.4) was added to another vial, and the samples were stored at 37℃ for one week. The stability changes of the samples were analyzed using a Formulaction multiple light scattering analyzer.

[0041] like Figure 4 As shown, after one week of storage, the fresh dUHT milk in the control group exhibited visible precipitation due to hydrolysis by fibrinolytic enzymes, which disrupted the milk's stable system. The dUHT milk supplemented with peptide YHX-PIP-2 remained stable after one week of storage.

[0042] The results of the stability analysis are as follows Figure 5 As shown, the TSI index of fresh dUHT milk (blank group) increased from 15 to 35 within 24 hours, while the TSI index of milk with added peptide YHX-PIP-2 was between 5 and 10, indicating that the plasmin inhibitory peptide YHX-PIP-2 of the present invention can effectively inhibit plasmin activity and extend the shelf life of dUHT milk.

[0043] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, alterations, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. The application of a β-lactoglobulin-derived plasmin inhibitory peptide in the preparation of food additives, characterized in that, The amino acid sequence of the β-lactoglobulin-derived plasmin inhibitor peptide is shown in SEQ ID NO.

1. The food additive is added to UHT milk to extend the shelf life of UHT milk.

2. The application of a β-lactoglobulin-derived plasmin inhibitory peptide in the preparation of hemostatic drugs, characterized in that, The amino acid sequence of the β-lactoglobulin-derived plasmin inhibitory peptide is shown in SEQ ID NO.1.

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